Nanoparticles for Photodynamic Therapy Applications

Peptide-conjugated nanoparticles for photodynamic therapy

targeting neuropilin-1

Pierre Couleaud1*, Denise Bechet3*, Régis Vanderesse2,5, Muriel Barberi-Heyob3,5, Anne-

Charlotte Faure4, Stéphane Roux4, Olivier Tillement4, Sabine Porhel1, François Guillemin3,5,

Céline Frochot1,5

1 Laboratoire Réactions et Génie des Procédés (LRGP), Nancy-University, CNRS, Nancy, France. 2 Laboratoire de Chimie Physique Macromoléculaire (LCPM), Nancy-University, CNRS, Nancy. 3 Centre de Recherche en Automatique de Nancy (CRAN), Nancy-University, CNRS, Centre Alexis Vautrin, Vandœuvre-lès-Nancy, France. 4 Laboratoire de Physico Chimie des Matériaux Luminescents (LPCML), UMR 5620 CNRS, Lyon I-University, Lyon, France 5 GDR CNRS 3049 "Médicaments Photoactivables-Photochimiothérapie (PHOTOMED)"

* contributed equally to this study

Correspondence to: Céline Frochot (for chemical and photophysical approaches) Laboratoire Réactions et Génie des Procédés 1 rue Grandville BP 20451 F-54001, Nancy, France Telephone: +33 3 83 17 51 15 [email protected] Muriel Barberi-Heyob (for molecular and cellular approaches) Centre de Recherche en Automatique de Nancy Centre Alexis Vautrin Avenue de Bourgogne, Brabois F-54511 Vandœuvre-lès-Nancy, France Telephone: +33 3 83 59 83 76 [email protected]

Couleaud P., Bechet D. et al., Nanomedicine, 2010.

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mailto:[email protected]

mailto:[email protected]

Keywords: peptide; functionalized-nanoparticles, neuropilin-1; in vitro photodynamic efficiency, molecular affinity Number of text pages, including references: 22 Number of figures: 7 Number of references: 70 Number of words in abstract: 119 Non-standard abbreviations: ABDM, 9,10 anthracenediyl-bis-(methylene)dimalonic acid; ADBA, disodium 9,10-

anthracenedipropionic acid; APTES, aminopropyltriethoxysilane; ATCC, American Type

Culture Collection; ATWLPPR, H-Ala-Thr-Trp-Leu-Pro-Pro-Arg-OH; a.i., arbitrary

intensity; a.u., arbitrary units; BSA, bovine serum albumine; BtOH, 1-hydroxybenzotriazole;

DEG, diethyl glycol; DLI, drug-light interval; DIEA, diisopropylethylamine; DMF,

dimethylformamide, DMSO, dimethyl sulfoxide; DPBF, 1,3 diphenylisobenzofuran; i.v.,

intravenous; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; FBS, fetal

bovine serum; Fmoc, 9-fluorenyl-methoxy-carbonyl; Gd2O3, gadolinium oxide; HBTU, 2-

(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate; HPLC, high

performance liquid chromatography; LC-MS, liquid chromatography-mass spectroscopy;

MRI, magnetic resonance imaging; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide; MWCO, molecular weight cut off; NaCl, sodium chloride; NP,

nanoparticle; NRP-1, neuropilin-1; Pbf, 2.2.4.6.7-pentamethyldihydro-benzofuran-5-sulfonyl;

PCS, photon correlation spectroscopy; PBS, phosphate-buffered saline; PDT, photodynamic

therapy; PEG, polyethylene glycol; PFP, pentafluorophenol; RGD, H-Arg-Gly-Asp-OH;

RNA, ribonucleic acid; RNO, N,N dimethyl-4-nitrosoaniline; SD, standard deviation; SOSG,

singlet oxygen sensor green; tBu, tertio-butyl; TEA, triethylamine; TEMP, 2,2,6,6-tetraethyl-

4-piperridone; TEOS, tetraethyl orthosilicate; TFA, trifluoroacetic acid; TLC, thin-layer

chromatography; TPC, 5-(4-carboxyphenyl)-10,15,20-triphenyl-chlorin; TPC-NHS,

5,10,15,tri-(p-tolyl)-20-(p-carboxylphenyl)chlorin succinidyl ester; TPP, tretraphenyl

porphyrin; UV, ultraviolet; VEGF, vascular endothelial growth factor; VTP, vascular targeted

photodynamic therapy; Φf, fluorescence quantum yield; 1O2, singlet oxygen; ΦΔ, 1O2 quantum

yield.


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Abstract

The strategy developed aims to favor the vascular effect of photodynamic therapy by

targeting tumor-associated vascularisation using peptide-functionalized nanoparticles. We

previously described the conjugation of a photosensitizer to a peptide targeting neuropilin-1

over-expressed in tumor angiogenic vessels. In this study, we have designed and

photophysically characterized a multifunctional nanoparticle consisting of a surface-localized

tumor vasculature targeting peptides and encapsulated photodynamic therapy and imaging

agents. The elaboration of these multifunctional silica-based nanoparticles is reported.

Nanoparticles functionalized with ~4.2 peptides specifically bound to neuropilin-1

recombinant protein. Nanoparticles conferred photosensitivity to cells over-expressing

neuropilin-1, providing evidence that the chlorin grafted within the nanoparticle matrix can be

photoactivated to yield photocytotoxic effects in vitro. Nanoparticles were eliminated by renal

excretion after intravenous injection.

Introduction

Photodynamic Therapy (PDT) is a treatment modality for cancer and various other diseases

[1,2]. This treatment involves the uptake of a photoactivable molecule called photosensitizer

by cancer tissue followed by a photoirradiation. Interaction of light with the photosensitizer

results in the formation of photosensitizer excited states that transfer its energy through

intermolecular triplet-triplet energy transfer to surrounding molecular oxygen (O2) molecules

and lead to the production of reactive oxygen species, such as singlet oxygen (1O2) [3,4].

Selectivity of PDT as an anti-cancer treatment relies on the local generation of cytotoxic

reactive oxygen species in the tumor and is achieved from the preferential uptake of the

photosensitizer by malignant tissue and from the subsequent localized light irradiation.

Several new strategies have emerged to improve the selectivity of PDT agents. Main of the

research is based on conjugation of vectors to target receptors over expressed on cancer cells,

such as oligonucleotides, monoclonal antibodies, epidermal growth factor, carrier proteins,

carbohydrates, folic acid for selective delivery of the agents into tumor tissues [5-8].

Destruction of the vasculature can also indirectly lead to tumor eradication, following

deprivation of life-sustaining nutrients and oxygen [9,10], and this vascular effect is thought

to play a major part in the destruction of some vascularized tumors by PDT [11-17]. Vascular

targeted photodynamic therapy (VTP) is a promising strategy and tumor-associated

vasculature a potential target of PDT damage.


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Synthetic peptides represent interesting targeting molecules, since they do not only exist in

natural form but can also be designed synthetically as novel molecules. In addition, the

effective tissue penetration of short synthetic peptides, in combination with their selective

binding and internalizing capacities by cancer cells, make peptides ideal candidates for the

delivery of therapeutic agents such as photosensitizers [18]. We have previously described the

conjugation of a photosensitizer (a chlorin) to an heptapeptide (ATWLPPR), specific for the

vascular endothelial growth factor (VEGF) receptor, neuropilin-1 (NRP-1) [19]. We

evidenced a statistically significant decrease of the ATWLPPR-conjugated photosensitizer

cellular uptake after RNA interference-mediated silencing of NRP-1 [20]. This targeted

photosensitizer proved to be very efficient in vitro in human umbilical vein endothelial cells

compared to its non-conjugated counterpart [19]. In vivo, we also demonstrated the interest of

using this active-targeting strategy by the induction of tissue factor expression immediately

post-treatment [21,22], and a specific localization in endothelial cells lining tumor vessels of

the conjugated photosensitizer [20]. Nevertheless, we could observe a degradation of the

peptide in plasma from 2 h post-intravenous injection and biodistribution results suggested

that its appearance in plasma mainly resulted from the degradation of the peptidic moiety into

organs of the reticulo-endothelial system [23,24].

As we previously described, non-biodegradable nanoparticles seem to be very promising

careers satisfying all the requirements for an ideal targeted PDT [25,26]. Hybrid gadolinium

oxide nanoparticles have been suggested to be useful for magnetic resonance imaging (MRI)

and treatment [27-29]; their small size (less than 50 nm of diameter), their silica shell that

allows the covalent coupling of a photosensitizer, their polyethylene glycol (PEG) grafting

that allows the covalent coupling of peptide units make them an ideal nanoplatform for both

imaging and VTP. In this targeting strategy using nanomaterials, we conjugated a second

generation photosensitizer, 5-(4-carboxyphenyl)-10,15,20-triphenyl-chlorin (TPC) and the

ATWLPPR heptapeptide to a hybrid gadolinium oxide nanoparticle. The paper focuses on its

functionalization, photophysical properties as well as cytotoxicity and photodynamic

efficiency in vitro, describing for the first time the molecular affinity of the functionalized

nanoparticle to NRP-1 molecular target.

Materials and Methods

The synthesis of gadolinium oxide nanoparticles embedded in a polysiloxane shell has been

widely described previously by our group [30-32].


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Peptide synthesis. Synthesis of the protected peptide H-AT(tertio-butyl (tBu))W(N-tert-

butoxy-carbonyl)LPPR(2.2.4.6.7-pentamethyldihydro-benzofuran-5-sulfonyl (Pbf))-OH was

carried out on a fully automated ResPepXL synthesizer (Intavis AG, Köln, Germany)

according to a classical 9-fluorenyl-methoxy-carbonyl (Fmoc)/tBu solid-phase methodology.

For the coupling of each amino acid, double couplings were performed using a threefold

excess of N-Fmoc-amino acid and activation reagents 2-(1H-benzotriazol-1-yl)-1,1,3,3-

tetramethyl-uroniumhexafluorophosphate (HBTU) (3 eq.), 1-hydroxybenzotriazole (BtOH) (3

eq.) and N-methymorpholin (9 eq.) in dimethylformamide (DMF). The cleavage, was realized

by a 2 h treatment with an acetic acid/trifluoroethanol/dichloromethane (2:2:6) mixture. The

protected peptide was eluted with a gradient of acetonitrile/H2O, 0.1 % trifluoroacetic acid

(TFA), from 40/60 to 90/10 for 20 min, followed by 100% acetonitrile for the last 15 min. A

preparative Pursuit C18, 5µm column (250 x 10 mm I.D., Varian, France) was used for

purification. Fluorescence detection was optimized with excitation and emission wavelengths

of 280 and 350 nm respectively coupled with an UV detection at 280 nm. Unless otherwise

stated, reagents were purchased from chemical companies and used without prior purification.

Fmoc-Arg(Pbf)-Wang and H-Arg(Pbf)-2-chlorotrityl resins, and Fmoc-amino acids were

from Merck Novabiochem. Thin-layer chromatography (TLC) was carried out on Merck

silica gel 60 F254 plates (Merck Chimie S.A.S., Fontenay sous Bois, France). The TLC spots

were visualized either by UV light or by heating plates sprayed with a solution of

phosphomolybdic acid (5% ethanol solution).

Photosensitizer synthesis. 5-(4-carboxyphenyl)-10,15,20-triphenylchlorin (TPC) was

synthesized as described by Whitlock [33] and our group [34] as well as 5,10,15,tri-(p-tolyl)-

20-(p-carboxylphenyl)chlorin succinidyl ester (TPC-NHS) [14].

Grafting of TPC-NHS into the polysiloxane shell. A solution containing TPC-NHS (6.2

mg) and 3.8 µL of aminopropyltriethoxysilane (APTES) dissolved in 1.30 mL of dimethyl

sulfoxide (DMSO) was prepared as a precursor and stirred overnight. The coupling reaction

between the chlorin and APTES was certified by mass spectrometry with complete

disappearance of the molecular peak of TPC-NHS (m/z= 758.1 (M-H+)) and the appearance

of the molecular peak of the chlorin conjugated APTES (m/z = 864.6 (M-H+), 1726.9 (2M-

H+) and 1747.3 (2M-Na+)). TPC-APTES solution and a portion of 327 µL of APTES and of

209 µL of tetraethyl orthosilicate (TEOS) were added to 1.5 mL solution containing Gd2O3


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nanoparticles diluted in 13.5 mL of diethyl glycol (DEG) under stirring at 40°C. After 1 h, a

portion of 792 µL of a DEG solution (0.1 M of triethylamine (TEA), 10 M of water) was

added. The other portions of polysiloxane precursors and of hydrolysis solution were

sequentially and alternatively added. The final mixture was stirred for 48 h at 40°C.

Covalent grafting of carboxylated PEG250 on hybrid nanoparticles. For grafting PEG250

via its carboxylic acid function (COOH), COOH functions were activated by 1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and pentafluorophenol

(PFP). 500 µL of DEG solution containing PEG250-COOH (0.091 M) were added to an

isopropanol solution (5 mL) containing a mixture of EDC/PFP (0.367 M). The reaction

between COOH groups of the polymer and PFP, assisted by EDC, is expected to yield

pentafluorophenyl esters. The resulting mixture was stirred for 90 min at room temperature

and added to 1 mL of colloidal suspension composed of hybrid nanoparticles (Gd2O3 cores

encapsulated in a polysiloxane shell) dispersed in DEG. The covalent grafting of PEG250-

COOH onto the hybrid nanoparticles was performed overnight at room temperature with

gentle stirring. The colloids of pegylated nanoparticles were introduced in a tubular

membrane of dialysis (molecular weight cut off (MWCO) = 4000-6000 Da) and immersed in

250 mL of water which was replaced four times every 12 h. Diluted purified solutions were

finally concentrated by ultrafiltration using Vivaspin® (MWCO = 10 000 Da).

Covalent grafting of the protected peptide on PEG250. 1.67 10-8 mol of Pegylated-

nanoparticles was dispersed in 7 mL dimethylformamide (DMF). The PEG carboxylic acid

functions were first activated by the classic peptidic synthesis mixture HOBt/HBTU/DIEA,

1/1/6 (HOBt 8.3 .10-8 mol, 12 µg, HBTU 8.3 .10-8 mol, 32 µg, DIEA 10 μL). The mixture was

stirred during 10 min before addition of 105 µg (8.3.10-8 mol) protected peptide. After 18 h

DMF was evaporated, 5 mL of pure TFA was added and the stirring was carried on during 2

h. Ultrapure water was added and the solvents evaporated. Several cycles of addition of water

and evaporation were realized to eliminate most of the TFA. The purification of the peptide-

functionalized nanoparticles was made by dialyses and ultrafiltrations. First dialysis was

realized with a 5000 Da cellulose membrane against ultrapure water. Several cycles of

dialysis were realized during 2 or 3 h each followed by ultrafiltrations with Vivaspin®

(MCWO 5000 Da) during 7 min at 3500 rpm. Finally, the water was removed under vacuum.


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https://www.researchgate.net/publication/41089707_Neuropilin-1_Targeting_Photosensitization-Induced_Early_Stages_of_Thrombosis_via_Tissue_Factor_Release?el=1_x_8&enrichId=rgreq-7f040f08-572f-47cb-b4d0-197b9cbcb858&enrichSource=Y292ZXJQYWdlOzUwNjAxMTE2O0FTOjEwMjkwODc1ODU5MzU0M0AxNDAxNTQ2NzY4Njc0

Size measurement. Direct measurements of the size distribution of the nanoparticles were

performed by photon correlation spectroscopy (PCS) with Zetasizer NanoZS from Malvern

Instrument. Measurements were taken on the colloids after purification by dialysis.

Zeta-potential measurements. Direct determination of the ζ-potential of the nanoparticles

were performed using a Zetasizer NanoZS (laser He-Ne (633 nm) from Malvern Instrument.

Prior to the experiment, the aqueous colloid was diluted in an aqueous solution containing

0.01 M NaCl and adjusted to the desired pH.

Photophysical properties. Absorption spectra were recorded on a Perkin-Elmer (Lambda 2,

Courtaboeuf, France) UV-visible spectrophotometer. Fluorescence spectra were recorded on a

SPEX Fluorolog-3 spectrofluorimeter (Jobin Yvon, Longjumeau, France) equipped with a

thermo stated cell compartment (25°C), using a 450 W Xenon lamp. Fluorescence quantum

yields (Φf) were determined using a tretraphenyl porhyrin (TPP) solution as a fluorescence

standard (Φf = 0.11, in toluene, taking into account solvent refractive index and absorption

efficiencies) [35]. For the direct determination of singlet oxygen quantum yield (ΦΔ):

excitation occurred with a Xe-arc, the light was separated in a SPEX 1680, 0.22 µm double

monochromator. The detection at 1270 nm was done through a PTI S/N 1565

monochromator, and the emission was monitored by a liquid nitrogen-cooled Ge-detector

model (EO-817L, North Coast Scientific Co). The absorbance of the reference solution

(Bengal pink in EtOH ΦΔ = 0.68 [36] and the sample solution (at 515 nm) were set equal

(between 0.2 and 0.5) by dilution. Determination of ΦΔ has been also realized with a chemical

probe, the singlet oxygen sensor green (SOSG, Invitrogen). The SOSG is highly selective for 1O2 [37] and can be used in aqueous solution. It initially exhibits weak blue fluorescence with

excitation peaks at 371 nm and 393 nm and emission peaks at 395 nm and 416 nm. In the

presence of oxygen, it emits a green fluorescence at 525 nm after excitation at 504 nm. SOSG

reagent was dissolved in methanol to make a stock solution of 5 mM. SOSG (final

concentration 50 µM) was added to the photosensitizer (2 mg mL–1) and 3 mL water was

added. Following excitation of SOSG at 480 nm using a 450 W Xenon lamp of the SPEX

fluorolog-3, the fluorescence was recorded at 525 nm.

Cell line and culture. To argue the involvement of NRP-1, MDA-MB-231 breast cancer cells

were used, strongly over-expressing NRP-1 receptor [38]. For in vitro experiments, MDA-


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MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas,

VA, USA). Cells were maintained in minimum essential medium (RPMI 1640) supplemented

with 10% fetal bovine serum (FBS), 1% antibiotic/antimitotic solution. They were cultured in

75 cm2 flasks and incubated in 5% CO2/95% humidified air at 37°C. Cell culture materials

were purchased from Costar (Dutscher, Brumath, France). All other chemicals were

purchased from Sigma (Saint Quentin Fallavier, France).

Binding of functionalized nanoparticles to recombinant NRP-1 protein. Neuropilin-1 was

obtained from R&D Systems (Lille, France), as recombinant chimeric protein. The surface of

Maxisorp microplates (Dutscher) was coated with NRP-1 (2 µg/mL) in phosphate buffered

saline (PBS), overnight at room temperature. The plates were blocked with PBS containing

0.5% bovine serum albumine (BSA, blocking buffer) during 1 h at 37°C, to prevent non-

specific interactions. Binding of the functionalized nanoparticles to NRP-1 was assessed

using 5 ng/mL of biotinylated VEGF165 (R&D Systems) in blocking buffer containing 2

µg/mL heparin. Biotinylated VEGF165 was added to the coated wells, in competition after an

excess of NP-TPC-ATWLPPR or control NP alone or unlabelled VEGF165 as positive control

(0.5 µg/mL, R&D Systems). After a 2 h-incubation at room temperature, the plates were

washed and the amount of bound biotinylated VEGF165 stained with streptavidin horseradish

peroxidase conjugate (R&D Systems) and assayed. After 20 min at room temperature,

reaction was stopped by the addition of Stop Solution (R&D Systems). Optical densities were

measured at 450 nm. Results were expressed as relative absorbance to wells containing only

blocking buffer. Three wells per condition were used. Reported values are the average of

triplicate measurements.

Dark cytotoxicity. Cell survival after incubation with the different batches of nanoparticles in

the dark was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

(MTT) assay. Briefly, MDA-MB-231 cells were plated at an initial cell density of 5 x 103

cells/well in 96-well microtitration plates and allowed to attach overnight. Wells were then

emptied, rinsed twice with PBS and filled with 200 µL RPMI medium containing various

concentrations of nanoparticles (photosensitizer concentrations ranged from 0.1 to 57.0 µM),

and the corresponding concentrations of gadolinium oxide (from 0.01 to 0.50 mM) for the

control nanoparticles. After a 24h-incubation at 37°C, wells were emptied, rinsed twice with

cold PBS and filled with 200 µL RPMI. Cell survival was measured 24 h later, as described

previously [39]. Experiments were carried out in triplicates.


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Photodynamic activity and light source. Photodynamic activity of nanoparticles on MDA-

MB-231 was also assessed by MTT assay. Cell survival was measured 24 h after

photosensitization. MDA-MB-231 cells were seeded at the initial density of 2 x 104 cellsmL-

1 in 96-well microtitration plates. Forty-eight hours after plating, cells were exposed to

photoactive nanoparticles at 0.1 and 1 µM. After 24 h incubation at 37°C, the medium was

removed, and cells were then washed three times with cold PBS and fresh RPMI was added

before cell irradiation. Irradiation was carried out at 652 nm, using a dye laser (Spectra-

Physics 375B, Les Ulis, France), pumped with an argon laser (Spectra-Physics 2020). The

output power was 700 mW. The light spot was 14 cm in diameter, providing a fluence rate of

4.54 mW/cm2. Light doses ranged from 1 to 20 J/cm2. During irradiation, the temperature

never exceeded 24±2°C. This temperature did not influence cell viability. Caution was taken

to avoid exposure of cells to light before and after irradiation. Negative controls were

photosensitizer free-medium without photo-irradiation, photosensitizer free-nanoparticles

(control NP) with photo-irradiation, photosensitizer containing-medium without photo-

irradiation. Experiments were carried out in triplicates at different days. Irradiation doses

yielding 50% growth inhibition (LD50) were determined using the medium effect method

[19].

Biodistribution of nanoparticles. Female NMRI nude mice (6–8 weeks old, Janvier, France)

were anesthetized (isoflurane/oxygen 4% for induction and 2% thereafter) and were injected

intravenously with a hybrid gadolinium oxide nanoparticles coated by PEG250 (200 µL)

containing Cy5 mono-NHS ester (Amersham Bioscience). The mice were illuminated by 633

nm light-emitting diodes equipped with interference filters. Fluorescence images, as well as

black-and white pictures, were acquired by a back-thinned charge-coupled device cooled

camera (ORCAII-BT-512G, Hamamatsu). A colored glass long-pass filter (RG 665) cut off

all excitation light in the image [40,41]. In vivo imaging experiments were reproduced at least

twice.

Statistical analysis. Mann-Whitney U test was used to assess the significant level between

independent variables. The level of significance was set to p < 0.05.

Results

Synthesis of nanoparticles, grafting with photosensitizer and peptide. Pegylated hybrid

gadolium oxide nanoparticles have been synthesized following several steps described in a


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previous study [28]. As also shown in other previous papers, the encapsulation of the

gadolinium core in a polysiloxane shell afforded the opportunity to enlarge the palette of

properties of the nanoparticles [27-29,32,42]. The functionalization of the nanoparticles was

rendered possible by the use of APTES whose hydrolysis-condensation in presence of TEOS

yields a polysiloxane shell anchored onto each oxide core. The amine function of APTES

allowed the immobilization of various molecules inside and/or onto the shell. The

modification of APTES before the hydrolysis induces the distribution of the conjugates inside

the shell whereas the derivatization of the polysiloxane shell will favors the distribution of the

conjugates onto the shell. The photosensitizer (TPC-NHS) was conjugated to APTES before

the hydrolysis-condensation whereas the ATWLPPR peptide units were immobilized onto the

nanoparticles after the derivatization of the polysiloxane shell by highly hydrophilic PEG250-

COOH chains. For the linkage of ATWLPPR to the nanoparticle, two pathways were possible

and described in Figure 1A. Figure 1B shows the chromatographic profiles of the nanoparticle

coupled to the peptide, the free peptide and the mixture of both. Some previous work [43]

proved that it was possible to use a wide pore C5 column with a gradient of water and

acetonitrile to separate gold nanoparticles (3 nm diameter) from proteins. In our case, after

several attempts with C4 and C18 column and different gradients, we concluded that the best

chromatographic separation was performed with a C18 column and a methanol/water

gradient. By this original and appropriated elution condition, it was possible to check that all

the free peptide has been eliminated after dialysis and ultrafiltrations.

Nanoparticles sizing and zeta potential. Size distribution and Zeta potential are given in

Figures 2A and 2B, respectively. Hydrodynamic diameter of the nanoparticles was comprised

between 8 and 14 nm (Fig. 2A) whereas the Gd2O3 cores have a hydrodynamic diameter of

3.8 nm (data not shown). Hence, encapsulation of the gadolinium oxide core into hydrophilic

shell containing fluorophore was reflected by the increase of the hydrodynamic diameters of

the particles. The nanoparticles exhibited a negative charge at pH 7.4 (Fig. 2B). As expected,

the derivatization of the nanoparticles by PEG250 rendered them water soluble in a wide pH

range, including pH of biological fluid whereas the colloidal stability of uncoated

nanoparticles was not sufficient. For pH>4, the negative charge was attributed both to the

negative charge of carboxylate groups, resulting from the deprotonation of COOH

(pKa(COOH/COO-) ~ 5), the negative charge of silanolate and from the steric hindrance and

hydrophilic character of PEG chains.


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Photophysical properties and singlet oxygen formation. Regarding the photophysical

properties, Figure 3A shows the absorption spectra of free TPC and different suspensions of

nanoparticles with or without peptide. No shift of the maximum absorption at 410 nm can be

observed, meaning that the photosensitizer was mainly in a monomeric form without

aggregation inside the nanoparticles. The low decrease of the nanoparticle fluorescence

emission indicated that the photosensitizer molecules linked into the silica matrix were

slightly affected by the micro environment (Fig. 3B). As illustrated in Figure 3C, the

signature phosphorescence emission of 1O2 at 1270 nm was clearly observed under 414 nm

excitation of the nanoparticles. Sensor green fluorescence increased rapidly after excitation of

the nanoparticles (Fig. 3D). No significant changes have been measured for free or coupled

TPC fluorescence lifetimes, fluorescence and singlet oxygen quantum yields (Fig. 3F).

Quantification of ATWLPPR grafting. Thanks to the fluorescence of the tryptophan residue

(excitation and emission wavelengths at 280 and 350 nm, respectively), it was possible to

estimate the number of grafted peptide onto the nanoparticles (Fig. 3D). The number of the

peptide units coupled to the nanoparticles has been estimated by comparing the fluorescence

of the tryptophan of the peptide coupled to the nanoparticle to a fluorescence calibration curve

of the peptide in solution. We found ~4.2 peptides per nanoparticles.

Molecular affinity of nanoparticles with peptides. As previously described, the endothelium-

homing peptide ATWLPPR, selectively targets to angiogenic vasculature and NRP-1 receptor

[19-23]. To assess the ability of ATWLPPR peptide to target nanoparticles to tumor-

associated vasculature, we synthesized and characterized multifunctional nanoparticles.

grafted with TPC and encapsulated gadolinium oxide within the core matrix. Polyethylene

glycol was attached to the surface of the nanoparticle along with the homing peptide. The

number of ATWLPPR peptide, when analyzed by spectrophotometry, revealed an average of

4.2 peptides per nanoparticles. The molecular affinity of the functionalized nanoparticles for

recombinant NRP-1 protein, has been estimated using binding tests. As VEGF165 binding to

its receptors is heparin dependent, the competitive binding experiments were always carried

out in the presence of heparin. Binding of biotinylated VEGF165 to NRP-1 was displaced by

the NP-TPC-ATWLPPR. Nanoparticles conjugated to ATWLPPR indeed bound to

recombinant NRP-1 chimeric protein without interaction of the nanoparticle alone (Fig. 4).

Interestingly, at 100 µM of peptides, we quantified for the functionalized nanoparticles (~4.2

peptides per nanoparticles) a better binding than the peptide alone.


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Dark cytotoxicity. As previously demonstrated, we used MDA-MB-231 breast cancer cells

strongly over-expressing NRP-1 receptor [38]. A MTT test was used to evaluate the dark

cytotoxicity of the different batches of nanoparticles (control NP, NP-TPC without peptide

and NP-TPC-ATWLPPR) for concentrations of TPC ranging from 0.10 to 57.00 µM. A 24 h-

incubation of MDA-MB-231 in the absence of light exposure with either photoactive

nanoparticle yielded a surviving cell fraction higher than 85% for concentrations up to 10.0

µM (Fig. 5). Control nanoparticles without chlorin displayed practically no cytotoxic effect

for all concentrations tested until 0.5 mM. We estimated an IC50 value of 16.9 µM for the

nanoparticles with TPC, corresponding to an equivalent molar concentration of 0.15 mM for

the control nanoparticles without photoactivable compound (Fig. 5). All subsequent

experiments were carried out at non-cytotoxic concentrations equal or inferior to 1.0 μM.

Photodynamic activity. MDA-MB-231 cells were incubated with the control nanoparticles

without TPC and peptide (NP), with TPC without peptide (NP-TPC), and with TPC and

peptide (NP-TPC-ATWLPR) and irradiated by red light (652 nm). Whereas NP-TPC at 0.1

µM displayed a weak photodynamic activity in MDA-MB-231 cells, conjugation with

ATWLPPR significantly enhanced photodynamic activity (Fig. 6A). The LD50 values of NP-

TPC and NP-TPC-ATWLPPR at 1µM of TPC were 5 and 3 J/cm2, respectively (Fig. 6C, D).

In vivo biodistribution. Figure 7 illustrates the biodistribution of nanoparticles at various

durations after the intravenous injection to nude mice. It appeared that only the kidneys and

the bladder, which are involved in renal excretion, were fluorescent as soon as 15 min after

the injection. Light emitted from the bladder vanished 1 h post-intravenous injection.

Discussion

The goals of this study were to synthesize, characterize and evaluate a multifunctional

nanoparticle formulation for use as a vascular-targeted photosensitizer delivery vehicle. We

found that the preparations of these nanoparticles could be readily achieved. For the linkage

of the ATWLPPR peptide to the nanoparticle, two pathways were investigated. The first one

involved the coupling of the nanoparticle on the preformed peptide linked to the resin. By this

way, the number of peptides per nanoparticle was not clearly determined as the large excess

of peptide, inherent to the synthesis conditions in term of scale, compared to the number of


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nanoparticles led to an overloading. Furthermore, after cleavage and deprotection, it was not

possible to disperse these overloaded nanoparticles into an aqueous medium. However, this

method might be useful with a great amount of nanoparticle. The other pathway relied on the

cleavage of the protected peptide from the resin, its purification followed by the coupling of

the selected amount on a defined quantity of nanoparticles. By this way, a well-established

ratio peptide/chlorin/gadolinium oxide can be selected. This strategy has been retained for the

coupling of about four peptide units by nanoparticle. An original HPLC method was

developed to check the complete elimination of all free peptide after dialysis and

ultrafiltrations.

The photophysical study of these nanoparticles with or without peptide grafting

revealed a preservation of the attractive photophysical characteristics of TPC such as the 1O2

quantum yield. It is accepted that 1O2 mechanism predominates during PDT and 1O2 is

generally believed to be the most important photocytotoxic specie. Different approaches have

been described to detect 1O2 produced by a photosensitizer encapsulated or covalently

attached into silica nanoparticles such as the use of chemical probes ADBA (disodium 9,10-

anthracenedipropionic acid) [44-46], DPBF (1,3 diphenylisobenzofuran) [25,47-50], ABDM

(9,10 anthracenediyl-bis-(methylene)-dimalonic acid) [51,52], RNO (N,N-dimethyl-4-

nitrosoaniline) with imidazole as a chemical trap [47,53], TEMP (2,2,6,6-tetraethyl-4-

piperridone) as a spin-trapping agent [52,54] or direct luminescence of 1O2 [25,26,44,45,50,

55,56]. In this study, we applied both direct luminescence of 1O2 and a chemical probe that

has been described to be selective to 1O2. Sensor green is a relatively new commercially

available reagent which is highly selective for singlet oxygen and can be used in water. It has

already been used in vivo for imaging the production of 1O2 [37]. A decrease of 1O2 quantum

yield was mentioned by others [53,57], suggesting that it can be due to indirect effects such as

the scattering of the nanoparticles, the local sequestration of generated 1O2 by the

nanoparticles matrix or intrinsic lower encapsulated photosensitizer 1O2 quantum yield. In our

case, sensor green fluorescence increased rapidly after excitation of the nanoparticles,

meaning that molecular oxygen can diffuse through the nanoparticles to react with the

chemical probe. Moreover, the ΦΔ do not decrease.

As the efficiency of PDT is mainly attributable to the production of 1O2, it is

unnecessary to release the loaded photosensitizers, but it is essential that O2 diffuses in and

out of the particles. Polyacrylamide polymers can be used for the synthesis of such


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nanoparticles, but most of them are ceramic-based or metallic. Their application in

photosensitizer delivery has not been fully exploited, even though the synthesis of this kind of

nanoparticle has been extensively reported in the literature [43]. In vitro characterization

studies of these nanoparticles revealed a photodynamic efficiency in a concentration-

dependent manner, providing direct evidence that the conjugated photosensitizer in the

nanoparticle matrix could be photoactivated to yield cytotoxic reactive oxygen species. Tada

et al. described the preparation and characterization of methylene-blue-containing silica-

coated magnetic particles. They showed that 1O2 generated by the photosensitizer

immobilized in the silica matrix also diffused. Nevertheless, the quantum yield was lower in

the silica-coated magnetic particles, estimated to be 3% versus 50% for the free methylene

blue [57]. Wieder and al. compared silica and gold nanoparticles, speculating that the surface-

bound photosensitizer on the gold nanoparticles might present a real advantage over an

encapsulated photosensitizer in terms of diffusion of 1O2 [58]. Conjugation with our hybrid

multifunctional nanoparticles did not induce significant variations in photophysical properties

and no changes have been measured between singlet oxygen quantum yields of TPC, NP-TPC

and NP-TPC-ATWLPPR.

A marked advantage of using non-biodegradable nanoparticles is that they can serve

as multifunctional platforms (For a review, see [25]). Kopelman et al. synthesized a

polyacrylamide multifunctional platform with a MRI contrast enhancer and Photofrin®, as

well as a vector (the integrin-targeting RGD peptide) for specific cell targeting [59-61]. In

vitro experiments confirmed the production of 1O2 at levels believed to be sufficient to induce

cell death. Applying a similar approach, Reddy and al. developed another polyacrylamide

nanoparticle encapsulating Photofrin® and imaging agents (fluorescent dye or iron oxide),

using an F3 peptide targeting surface-localized vasculature [62]. In our targeting strategy,

nanoparticles conferred photosensitivity to cells, providing evidence that the chlorin grafted

within the nanoparticle matrix can be photoactivated in vitro. The improved photodynamic

efficiency of peptides-conjugated nanoparticles compared to unconjugated nanoparticles, was

consistent with the binding to NRP-1 receptor followed by an active transport mechanism.

The molecular affinity was tested for functionalized nanoparticles, demonstrating the ability

of ATWLPPR homing heptapeptide to bind recombinant NRP-1 protein testing about four

peptides per nanoparticle. Peptide-conjugated chlorin demonstrated a molecular affinity of

213 µM (IC50 value) for NRP-1 recombinant chimeric protein but to a lesser extent than non-


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conjugated peptide [38]. The presence of four peptides ligand per nanoparticle seems to lead

to a positive cooperativity in binding of ATWLPPR to NRP-1 protein.

The potential of the multifunctional nanoparticles can be fully exploited for PDT applications

only if they do not non specifically accumulate, and if they are rather quickly eliminated.

Choi et al. emphasized the necessity of rapid clearance and pointed out the problems related

to accumulation of nanoparticles in vivo [63]. A rapid clearance of nanoparticles such as

quantum dots was observed only when their hydrodynamic diameter was smaller than 5.5 nm.

However, non specific accumulation of cysteine-coated quantum dots can be observed in the

liver even for the smallest particles, while the largest ones are mostly accumulated in the

liver, spleen, and generally ascribed to uptake by the reticulo-endothelial system [64]. The

detection of nanoparticles in the lungs seems to provide from their agglomeration caused by

the adsorption of plasma proteins [65]. The functionalization of nanoparticles by a PEG

coating is probably the most efficient strategy to avoid nonspecific accumulation, which could

be detrimental for the in vivo application. Many studies have indeed demonstrated the positive

effect of PEG on the biodistribution of such particles [66]. Derivatization by PEG chains

limits the uptake by resident phagocytes in the liver and in the spleen [67]. Because of steric

shielding, the PEG chains avoid the adsorption of plasma proteins onto the surface of the

nanoparticles (opsonisation), occurring when nanoparticles are injected into the bloodstream.

However, the effect of PEG chains on the biodistribution can be counterbalanced by the

charge of the particle. Although pegylation seems to induce prolonged blood circulation, in

some cases, it does not prevent the liver uptake of nanoparticles [69,70]. Our group also

investigated the influence of the length of different PEG on the biodistribution of hybrid

gadolinium oxide nanoparticles [28]. Hybrid gadolinium oxide nanoparticles coated by

PEG250 leaded a rapid in vivo elimination of the nanoparticle. Ex vivo examination of the

organs 24 h post-intravenous injection checked that the nanoparticles did not accumulate into

organs other than the kidneys, which remained strongly fluorescent. In order to preserve this

biodistribution characteristic, we only selected multifunctional nanoparticles describing few

peptides per nanoparticle. We previously evidenced that the peptide-conjugated

photosensitizer was mainly concentrated into organs of the reticulo-endothelial system,

leading to a degradation of the peptide moiety characterized by an enzymatic cleavage of the

peptide bond between Ala and Thr [20]. Interestingly, using functionalized nanoparticles

eliminated by renal excretion will hence overcome this degradation process.

This study revealed the photodynamic efficiency of multifunctional nanoparticles for

PDT. According to our in vitro results ATWLPPR-functionalized nanoparticles could not


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only target NRP-1 protein and cells over expressing NRP-1 but also in vivo endothelial cells

lining tumor vessels. This experiment is currently in progress.

Acknowledgements

This work was supported by the research funds of the French Ligue Nationale Contre le

Cancer and the ANR project no. ANR-08-PCVI-0021-01 Nano-VTP.


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Legends

Figure 1. Strategies of synthesis and purification of the targeted nanoparticles. (A) Two

possible pathways for the linkage of ATWLPPR to the nanoparticle. i) coupling of the

nanoparticle on the growing peptide attached to the resin (pathway A); ii) cleavage of the

protected peptide from the resin followed by coupling on the nanoparticle (pathway B); (B)

Reversed phase HPLC chromatogram of nanoparticles coupled to ATWLPPR (a), free peptide

(b), and mixture of both (c). λ ex =280 nm, λ em = 350 nm.

Figure 2. (A) Size distribution determined by photon correlation spectroscopy of hybrid

gadolinium oxide nanoparticles coated by PEG250. (B) Evolution of the ζ potential versus pH

for these nanoparticles.

Figure 3. Photophysical properties of free photosensitizer (TPC) and after its coupling to the

nanoparticles. (A) Absorption spectra of free TPC, free peptide and suspension of

nanoparticles with or without peptide in ethanol. (B) Fluorescence emission spectra of the free

TPC, and suspensions of nanoparticles with or without peptide in ethanol, for the same

absorbance (λexc=414 nm). (C) Singlet oxygen emission spectra of the free TPC, and

suspensions of nanoparticles with or without peptide in ethanol, for the same absorbance,

after excitation at 414 nm. (D) Fluorescence emission spectrum of the tryptophan residue

using suspensions of nanoparticles with peptide in ethanol (excitation and emission

wavelengths at 280 nm and 350 nm, respectively). (E) Fluorescence emission of sensor green

in water at 525 nm after excitation at 504 nm. Measurements were performed for 20-seconds

period, with 30-seconds interval between each measurement. (F) Fluorescence lifetimes and

quantum yields of fluorescence and singlet oxygen.

Figure 4. Binding of nanoparticles without ATWLPPR (NP-TPC, grey), with peptide

corresponding to an average of four molecules of ATWLPPR peptide per molecular of

nanoparticles (NP-TPC-ATWLPPR, black) to recombinant NRP-1 protein compared to

ATWLPPR alone (white). Binding of biotinylated VEGF165 (5 ng/mL; 110 pM) to NRP-1 in

the presence of 2 µg/mL heparin was evaluated when increasing concentrations of NP-TPC,

NP-TPC-ATWLPPR or ATWLPPR were added (data points show the mean ± SD, n=3).


17

Figure 5. Dark cytotoxicity of nanoparticles with (black) and without photosensitizer

(orange) in MDA-MB-231 cells, according to concentration, as determined by MTT test (data

points show the mean ± SD, n=6).

Figure 6. Measurements of photodynamic therapy sensitivity to control nanoparticles (NP,

white), nanoparticles with TPC without peptide (NP-TPC, grey) and nanoparticles with TPC

and functionalized with ATWLPPR (NP-TPC-ATWLPPR, black) in MDA-MB-231 cells.

Cells incubated with different concentrations of nanoparticles for 24 h before light treatment.

Survival curves were obtained for cells exposed to increasing doses of light from 1 to 20

J/cm² by MTT test (data points show the mean ± SD, n=6).

Figure 7. (A) Fluorescence reflectance imaging of a nude mouse lain on its back and (B) on

its belly after intravenous injection in the caudal vein of hybrid gadolinium oxide

nanoparticles. Each image was acquired with an exposure time of 200 ms.

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Nanoparticles for Photodynamic Therapy Applications

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